Efficient One-Pot Solvothermal Synthesis and Characterization of Zirconia Nanoparticle-Decorated Reduced Graphene Oxide Nanocomposites: Evaluation of Their Enhanced Anticancer Activity toward Human Cancer Cell Lines

This study mainly deals with an effective one-pot solvothermal synthetic pathway for the preparation of uniformly dispersed zirconium oxide nanoparticles on the flattened rough surface of reduced graphene oxide (ZrO2/rGO NCs) using the aqueous leaf extract of Andrographis paniculata. After obtaining detailed information on the preparation and characterization, the anticancer activity of the synthesized ZrO2/rGO nanocrystals (NCs) was evaluated on two human cancer cell lines (A549 and HCT116) along with one normal human cell line (hMSC). The 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide assays revealed that ZrO2/rGO NCs exhibited a dose-dependent cytotoxicity pattern. The cell viability (%) drastically decreases up to 96–98% after exposure to an optimal concentration of 10 ppm nanocomposites. Analysis of both the reactive oxygen species generation and the Annexin V-FTIC staining assays reveal that ZrO2/rGO NCs have the ability to induce apoptosis in A549 and HCT116 cell lines. Thus, the green synthesis of ZrO2/rGO NCs shows potential in developing efficient therapeutic agents for cancer therapy.


XRD Studies:
X-ray diffraction studies were employed to identify the crystal structure and determining the diffraction pattern of all the synthesized nanocomposites such as GO, rGO-AP, ZrO2/rGO NC of 0.1 0.05, 0.01and ZrO2 NP are represented in Figure S1A, where GO formation was confirmed with diffraction patterns at 10.25° and 42.30°, which were attributed to the d002 plane of GO and 100 carbon planes of a hexagonal structure, respectively ( Fig. S1A (a)). 1 The identified d-spacing values of GO were 8.62 A° and 2.13 A° respectively. After complete reduction of GO to rGO-AP, there was a significant reduction in the diffraction intensity was observed and moreover, two new diffraction peaks were identified at 25.63° and 42.96°, which correspond to the rGO-AP d002 plane with a d-spacing values of 3.41 A° and 2.09 A° respectively. This pattern confirms the successful reduction of GO to rGO and interestingly, the d-spacing was reduced from GO to rGO, the reason might be the removal/loss of oxygen functionalities during the reduction of GO ( Fig. S1A (b)). Also, the tetragonal-ZrO2 NPs formation was further confirmed with the observed diffraction pattern at 30.2 (101), 35.17 (110), 50.6 (200), 60.3 (211), and 74.6 (220) A⁰ and this pattern was well-matched with the JCPDS card no: 88-1007. All these diffractions of ZrO2 were observed in nanocomposites, however there were no rGO peaks found in the composite pattern, this might be due to the S3 uniform distribution of ZrO2 in between the graphene layers and absence of rGO influence on the crystalline phase growth pattern of ZrO2 during the calcination of composite. 2

FT-IR spectroscopy studies:
FT-IR analysis result reveals the functional groups existed on the surface of the nanomaterials. Fig. S1B represents the FT-IR spectra of GO, rGO-AP, and ZrO2/rGO NCs

DLS and Zeta potential studies:
It is well known that nanocomposites often have the ability to form the agglomerates in solution mediums, which shows further effect on their interactions with biological systems.
Thus, the interaction behaviour of the above synthesized samples GO, rGO-AP, ZrO2/rGO NC 0.01M, ZrO2/rGO NC 0.05M, ZrO2/rGO NC 0.1M, and ZrO2 NP 0.05M when exposed to three different liquid environments like PBS, culture medium and water systems were tested using DLS (dynamic light scattering) analysis. By using this analysis, we determined the hydrodynamic diameter, zeta potential and polydispersity indices (PI) values of different suspensions as illustrated in Table S1 and Figure (Table S1). Consistently, the zeta potential is decreased in PBS and cell culture medium (Table S1). This phenomenon is mainly due to the cell growth medium was composed of many biological components like high protein content of serum, albumin, fibrinogen, amino acids, vitamins, and also the presence of phosphate have each previously been shown an impact on agglomeration/precipitation of NPs. These findings serve better to S5 identify the cause for the decrease in colloidal stability upon particle incubations in biological mediums such as in PBS and DMEM medium. Polydispersity indices (PI) is an autocorrelation function, which corresponds to the ratio of second moment and square of mean value of logarithm and it denotes the extent of size distribution. 6 The ZrO2/rGO NC suspensions shows larger PI values with larger ionic strengths i.e., in DMEM (culture medium) and PBS when compared to water medium exhibiting lower ionic strengths (Table S1)  Figure S2. Hydrodynamic size distribution and Zeta potential of GO (0.5 mg mL -1 ) in PBS medium (1,4), Cell culture medium (2,5) and Water medium (3,6) were measured by DLS at room temperature. Figure S3. Hydrodynamic size distribution and Zeta potential of rGO-AP (0.5 mg mL -1 ) in PBS medium (7, 10), Cell culture medium (8, 11) and Water medium (9, 12) were measured by DLS at room temperature.    Abbreviations: Q1: necrotic cells, Q2: late apoptotic, Q3: region denotes live cells, and Q4:

S8
apoptotic. Table S6A. Statistical analysis of data sets for significant evaluation of anticancer activity of ZrO2/rGO NCs on A549 cell lines by MTT assay S16 S17 Table S6B. Statistical analysis of data sets for significant evaluation of anticancer activity of ZrO2/rGO NCs on HCT116 cell lines by MTT assay S18 Table S6C: Statistical analysis of data sets for significant evaluation of anticancer activity of ZrO2/rGO NCs on hMSCs cell lines by MTT assay